Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.


March 2021
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Characteristics of patients and healthy donors are detailed in Supplementary Tables 1-6 of the manuscript.
The main recruitment criterium was a confirmed diagnosis of myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndrome or secondary acute myeloid leukemia (sAML). As specified in the methods section, biosamples of MN patients were obtained from residual material from diagnostic BM aspirations. The study consisted of n=56 patients with myeloid neoplasms (MN), who were treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 72.5 years old, range 44-88). As for healthy controls (n=16), hematopoietic cells were obtained from bone specimen from femur endoprosthesis surgery (median age 67 years old, range 51-92). Bone marrow (BM) samples from these patients or healthy controls were used for RT-qPCR, CellTiter-Glo cell viability assay, LOX/LOXL activity inhibition assay, collagen production assessment, co-cultures of mesenchymal stem cell (MSC)/extracellular matrix (ECM) and hematopoietic stem and progenitor cells (HSPC) as well as patient-derived xenograft (PDX) models.
In the analysis of the concentration and enzymetic activity of LOX and LOXL2 in BM plasma, n=94 MN patients (median age 73 years old, range 43-88) and n=15 healthy donors (median age 25 years old, range 21-79) were included. These MN patients were also treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany. The BM of young healthy donors (<50 years old) was collected by voluntary iliac crest puncture. The BM from old healthy donors (>50 years old) were obtained as described above. All patients and healthy donors provided written informed consent and all interventions were performed in accordance with the Declaration of Helsinki. There was no bias in selection of patient samples.
The use of primary human materials for research purposes was approved by the Medical Ethics Committee II of the Medical Faculty Mannheim of the Heidelberg University.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
The sample size of involved human biosamples was not pre-determined. Sample size was based on the availability of patient cryopreserved samples stored in the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany. More detailed information of sample size is displayed in supplementary tables 1-6 of the manuscript.
In RT-qPCR, we included n=20 MN patients and n=9 healthy donors.
In the analysis of the concentration and enzymetic activity of LOX and LOXL2 in BM plasma, n=94 MN patients and n=15 healthy donors were included.
In the CellTiter-Glo cell viability assay, samples from n=11 MN patients and n=3 healthy donors were included. In the LOX/LOXL activity inhibiting assay, MSCs from n=5 MN patients were included.
In the co-cultures of MSC and HSPC and all colony-forming unit assays, we included a total of n=31 MN patients and n=7 healthy donors.
In the PDX models, MSCs and HPSCs from n=6 MN patients were used. A total of n=63 NSG mice were used for PDX model.
The sample size of n=15 animals per treatment group for the experiment with Wistar Han rats was required to reject the null hypothesis in ANOVA analysis of 4 groups with a power of 0.80 (type II error #=0.2) and type I error "=0.05.
There was no data exclusion.
For the experiments in this study, all attempts at replication were successful: In-vitro MSC/HPSC co-culture assays included the following readouts: blinded manual assessment of HSPCs colony formation in colony-forming unit (CFU) assays, flow cytometry assessment of erythroid, myeloid and megakaryocytic differentiation as well as assessment of erythroid progenitors clonality using panel sequencing. For some of MSC/ HSPC co-culture or MSC/extracellular matrix culture assays, we counted cell number of HSPCs after co-culture and treatment. These readouts were performed using at least n=3 biological replicates.
In-vivo PDX mice were grouped into untreated, 5-AZA, PXS-5505 and 5-AZA+PXS-5505 (P+A) arms. Totally, untreated arm included n=14 PDX mice, 5-AZA arm included n=17 mice, PXS-5505 arm included n=17 mice, and P+A arm included n=15 mice. In summary, we analyzed a sufficient number of MN samples and PDX mice to obtain statistical significance and ensure that the results were reproducible.
The effects of the PXS-5505 + 5-AZA combination were studied in patients with myelodysplastic and myeloid malignancies. Synergistic effects of the combination treatment on the erythropoiesis were observed in 11/31 = 35% of patients. To replicate the effects of the combination treatment for each responder or non-responder, critical in vitro experiments were performed using at least n=3 biological replicates (  Fig. 2d). The effects of the combination treatment on the cross-linked collagen production were assessed in n=2 patients with bone marrow fibrosis (Fig. 1k) and reproduced in n=3 co-culture assays ( Supplementary Fig. 2f).
Wistar Han rats were randomized on a weight stratified basis using Provantis TM 9.3.1, so that comparable distribution of body weights among groups was achieved after randomization (within ±20% of the mean). Patient samples for PDX studies (n=6) were selected based on the availability of the primary material and successful engraftment in NSG mice (>1% engraftment in the bone marrow before treatment start). For the experiments shown in Figures 6-8 only erythroid responders with sufficient amount of a primary material were selected based on the data shown in Fig. 2e.
We performed blinded manual assessment of HSPCs colony formation in CFU assays. The percentage of squares (tiles) containing the areas of fibrosis or intra marrow ossifications based on Gomori silver impregnation staining were calculated using QuPath software by the operator blinded to treatment groups. For other experimental assays with objective experimental readout (flow cytometry, LOX/LOXL activity and viability assays) data analysis was performed by an operator blinded to treatment groups. While evaluating the responses of the animals, the scientists were aware of the treatment history. However, due to the technical objectivity of the endpoints to be examined using instumental methods (clonal evolution using NGS sequencing, engraftment rates and erythroid differentiation by flow cytometry, spleen and body weights etc), it was not possible to introduce a bias because instrument setting were kept the same for all samples. Note that full information on the approval of the study protocol must also be provided in the manuscript. Patient-derived xenograft (PDX) models were established in 8-10 week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) female mice (Jackson Laboratory, 005557). All NSG mice were housed under specific pathogen-free conditions in an animal facility (Medical Faculty Mannheim, Heidelberg University) at a 12h/12h day/night cycle in individually sterilized ventilated plastic cages with adjusted air temperature (21°C) and 50% relative humidity. All NSG mice were fed ad libitum with a sterilized standard redent diet and free access to sterilized water. For the PXS-5505 toxicity studies in Wistar Han rats, both female and male rats were used at the age of 28-32 weeks. Rats were acclimated at Pharmaron animal facility for 3 weeks prior to treatment. Rats were housed in polycarbonate shoebox cages with corn cob bedding and 12-hour light/12-hour dark cycle. The temperature was set to be maintained between 20-26°C. The humidity was maintained between 40-70%. Rats were fed ad libitum with rodent diet provided by Beijing KeAo Xieli Feed Co., Ltd. Water was provided ad libitum via water bottles. The tap water was filtered through four sequential filters (5-10 $m, 1-4.9 $m, 0.2-0.5 $m, and 0.1-0.19 $m), passed through the ultraviolet sterilization system and filled into the water bottle. The bottle including water was autoclaved before given to the animals.
No wild animals were used in this study.
PDX models were performed in female NSG mice, as they are known to show higher success rate of obtaining human engraftment compared to males. For the PXS-5505 toxicity studies in Wistar Han rats the toxicity tests were performed on both females and males and toxicity data were reported separately for both sexes.
The study did not involve any sample collected from the field.